The present invention relates generally to in situ hybridization methods and hybridization buffers for labeling of interphase nuclei. More particularly, the present invention relates to a protocol and buffer which is particularly useful in conjunction with synthetic oligonucleotide probes for hybridizing DNA or RNA sequences by fluorescence in situ hybridization ("FISH"). In accordance with the preferred embodiments of the present invention, the inventive FISH protocol employs synthetic oligonucleotide probes having less than about 75 bases, preferably between about 15-50, most preferably between about 25 and 35 bases, and a single solution denaturation and hybridization buffer solution which permits the entire FISH protocol to be completed within time periods suitable for clinical diagnostic application. The present invention has been found particularly useful in fluorescence labeling of chromosomes X, Y, 13, 18 and 21 in human lymphocytes and amniocytes using synthetic oligonucleotide probes. Additionally, the present invention includes a methodology for conducting multiple, repeat rounds of FISH analysis, using different probe sets on the same sample. This repeat FISH methodology ("ReFISH") permits a sample to be subjected to a first FISH analysis with one probe then subsequently subjected to FISH analysis with a different probe. In all aspects of the present invention, the criteria for success included: 1) ease of procedure, 2) reliability, 3) hybridization specificity, 4) hybridization efficiency, 5) brightness of the fluorescence signal, 6) stability of the labeled hybrid, and 7) conservation of the cellular and nuclear morphology.
Conventional FISH technology is based on formamide chemistry for DNA denaturation, hybridization buffers, and post-hybridization washes. (Cremer T. Landegent J. Brueckner A, Scholl H P, Schardin M, Hager H D, Devilee P. Pearson P. van der Ploeg M. (1986), "Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and non-radioactive in situ hybridization techniques: diagnosis of trisomy 18 with probe L .84," Hum. Genet 74:346-352; Pinkel D, Straume T. Gray J W. (1986) "Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization," Proc Natl Acad Sci USA 83:2934-2938). The entire procedure includes multiple steps for sample denaturation, hybridization and post-hybridization washes. This process often takes hours to yield a fluorescent label with a signal intensity sufficient for routine chromosome enumeration. Although some recent improvements of formamide-based FISH have been introduced, for example the use of co-denaturation of probes and sample and the elimination of formamide in the post hybridization washes (Abati A, Sanford J. Fetsch P. Marincola F. Wolman S. (1995) "Fluorescence in situ hybridization (FISH): a user's guide to optimal preparation of cytologic specimens," Diagnostic Cytopathology 13:5:486-492), the chemistry of the hybridization reaction is still based on formamide. Known disadvantages of formamide are that it reduces the kinetics of hybridization (Kourilsky Ph. Leidner J. Tremblay. 1971 "DNA-DNA hybridization on filters at low temperature in the presence of formamide or urea,. Biochimie 53:1111-1114), oxides easily, and is a known teratogen.
Currently, most clinical laboratories use FISH kits available from the following commercial sources: Oncor, Inc., Rapid Chromosome In Situ Hybridization System: Edition 1, October 1993, or Vysis, Inc. Both the Oncor and the Vysis probes are biological probes and the kit protocols employ formamide-based hybridization solutions. Another current kit for conducting FISH is available from Aprogenex, Inc. and is sold under the trademark APROPROBE PLUS. The APROPROBE PLUS FISH kit utilizes synthetic oligonucleotide fluorophore-labeled probes specific for the centromeric region of human chromosomes X, Y, 13/21 and 18 and the mRNA of gamma globin. The hybridization solution provided with the APROPROBE PLUS FISH kit is also formamide-based. See, also, Bresser, et al., U.S. Pat. No. 5,225,326, which teaches the use of formamide-based hybridization solution with oligonucleotide probes.
Non-formamide-based hybridization solutions for conducting FISH analysis are known in the art, as more fully disclosed in co-pending U.S. patent application Ser. No. 08/418,704, filed Apr. 7, 1995, published as International Application WO 96/31626, published on Oct. 10, 1996, which discloses use of a glycerol and dextran sulfate hybridization buffer for FISH labeling of human chromosomes using clone probes. However, when the teachings of International Application WO 96/31626, were followed using synthetic oligonucleotide DNA probes, such those available in the APROPROBE PLUS FISH Kit (Aprogenex, Inc.) or those disclosed in International Application 96/00234, published Jan. 4, 1996, clinically adequate labeling of the human chromosomes was not obtained. International Application WO 96/31626, published Oct. 10, 1996, is hereby incorporated by reference as to the teaching of a glycerol-based hybridization solution and the use of a glycerol-based hybridization solution with biological DNA probes. International Application 96/00234, published Jan. 4, 1996, is hereby incorporated by reference as teaching synthetic oligonucleotide clone probes specific for human chromosomes X, Y, 13, 18 and 21 useful with the present invention.
It was found necessary, therefore, to modify the glycerol-based hybridization methodology taught in International Application WO 96/31626, to adapt it for use with synthetic oligonucleotide DNA probes. Specifically, it was found that reducing the hybridization temperature from 55.degree. C. to between 37 and 47.degree. C., changing the sodium ion concentration in the wash, and reducing the wash temperature from 65.degree. to a temperature which was substantially the same as the hybridization temperature, i.e., between 37 and 47.degree. C., yielded acceptable specific binding and retention of the fluorophore labeled synthetic oligonucleotide probe to human chromosomes X, Y, 13, 18 and 21 in lymphocytes, amniocytes, and metaphase chromosomes. In accordance with the best mode of the present invention, however, it was found that while the bound fluorescence signal was acceptable, the signal was not conserved sufficiently to achieve an acceptable duration of the fluorescence signal. It was further discovered that the addition of dithiothreitol to the hybridization buffer achieved a prolonged duration of the bound fluorescence signal and permitted the labeled samples to be archival stored and analyzed at a later time.
For each of the inventive FISH protocols, the chromosome-labeling process was completed within 90 minutes. The inventive processes are based upon a unique formamide-free hybridization chemistry that accelerates the probe-to-target annealing reaction without compromising hybridization efficiency, specificity, or cellular and nuclear morphology. The inventive FISH processes use synthetic oligomeric probes with sequences derived from the alpha satellite regions of human chromosomes X, Y, 13, 18, and 21. Hybridization specificity and efficiency, and chromosomal target were retained through the repetitive FISH processing on the same sample. The inventive formamide-free FISH chemistry is readily adapted for use with different cell types. The simple and rapid methods developed with this chemistry work with either lymphocytes, uncultured amniocytes or with metaphase chromosomes, and with cells fixed by different methods. The inventive ReFISH process introduces the possibility of FISH analysis for multiple chromosome targets with a sample that is small or otherwise limited by generating simple patterns for each analysis, without the need to have complex mixtures of different probes. This avoids the interpretation of more complex signal patterns when multiple-target probe mixtures are used in a single reaction. Another advantage of ReFISH is that archived samples can be reexamined by FISH at a later date, i.e., several weeks or even months later, either for re-confirmation or with another probe set that was not requested or available at the time of initial testing.